Inhibition of peroxidase enzymatic activity

ABSTRACT

The present invention deals with reagents and compositions capable of effectively inhibiting peroxidase activity. According to the invention, peroxidase enzymatic activity is blocked with an acidic aqueous solution of a protein denaturing agent. Preferred protein denaturing agents are detergents and chaotropic substances.

RELATED APPLICATIONS

This application claims priority to European application EP 06011949.2filed Jun. 9, 2006, now EP Patent No. 1865054, and U.S. patentapplication Ser. No. 11/758,854 filed Jun. 6, 2007, now U.S. Pat. No.8,735,083.

FIELD OF THE INVENTION

The present invention deals with reagents and compositions capable ofeffectively inhibiting peroxidase activity. In particular, the inventionrelates to blocking peroxidase enzymatic activity with an acidic aqueoussolution comprising a protein denaturing agent, in particular, adetergent or chaotropic substance.

BACKGROUND

A peroxidase is an enzyme, which may contain heme, that catalyzes areaction of the form:ROOR′₊electron donor (2e′)+2H⁺→ROH+R′OH

For many of these enzymes the optimal ROOR′ (electron acceptor)substrate is hydrogen peroxide, but others are more active with organichydroperoxides such as lipid peroxides.

The nature of the electron donor is very dependent on the structure ofthe enzyme. For example, horseradish peroxidase (HRP), which is isolatedfrom horseradish (Armoracia rusticana) roots, can use a variety oforganic compounds as electron donors and acceptors.

Being a member of the ferroprotoporphyrin group of peroxidases, HRP doescontain a heme group. Located at positions distal and proximal to theheme plane there are in addition two calcium binding sites. HRP Cdominates quantitatively among the isoperoxidases of horseradish root.HRP C is a single chain polypeptide comprising 308 amino acid residueswhich form four internal disulfide bridges and 8 neutral carbohydrateside-chains. The molecular weight of the polypeptide chain is 33890Daltons (Da), and the molecular weight of native horseradish peroxidaseC is about 44 kDa (Welinder, K. G., Eur. J. Biochem. 96 (1979) 483-502).At least seven isozymes of HRP exist (Shannon, L. M. et al., J. Biol.Chem 241 (1966) 2166-2172). The carbohydrate composition consists ofgalactose, arabinose, xylose, fucose, mannose, amnnosamine, andgalactosamine, depending upon the specific isozyme (Shannon, L. M., etal. J. Biol. Chem. 241 (1966) 2166-2172). The isoelectric point of theisozymes ranges from 3.0 to 9.0. The pH optimum of HRP is in the rangeof pH 6.0 to pH 6.5; activity at pH 7.5 is 84% of the maximum. Theenzyme is most stable in the range of pH 5.0 to pH 9.0 (Schomburg, D.,et al., Enzyme Handbook 7 (1994) EC 1.11.1.7:1-6).

HRP combines with hydrogen peroxide (H₂O₂) and the resultant [HRP-H₂O₂]complex can oxidize a wide variety of chromogenic hydrogen donors. HRPhas a broad and accessible active site and many chemically verydifferent compounds can reach the site of the reaction. Known HRPsubstrates include TMB (3,3′,5,5′-tetramethylbenzidine), ABTS(2,2′-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid diammonium salt),luminol (5-amino-1,2,3,4-tetrahydrophthalazin-1,4-dion) and isoluminol(4-aminophthalhydrazide), as well as fluorogenic substrates such astyramine (4-hydroxy-phethylamine), homovanillic acid, and4-hydroxyphenyl acetic acid. Further HRP substrates are known to theart. Due to its versatility, HRP is commercially used as a component ofimmunoassays, such as coupled enzyme assays, chemiluminescent assays andassay kits for clinical diagnostics including histochemistry kits.

In a typical example, an immunoassay based on a sandwich ELISA principleand using analyte-specific coating and capture antibodies includes HRPconjugated to the capture antibody. The peroxidase enzyme catalyzes thecleavage of a chromogenic substrate to yield a product which can bemeasured spectrophotometrically. The absorbance of a colored orfluorescent product is directly correlated to the amount of analyte inthe sample analyzed. In order to allow the comparison of simultaneousmeasurements including controls, the HRP enzymatic reaction needs to beterminated after a defined incubation period. To this end a stop reagentis used. Stop reagents have to fulfill two major requirements: (1) toterminate the reaction by effectively inhibiting the enzymatic activityof HRP; (2) to stabilize the oxidized products of the chromogenic orfluorogenic substrate(s).

In Zollner, H., Handbook of Enzyme Inhibitors, 2^(nd) Ed. (1989) Part A:367-368) the following compounds have been described as inhibitors ofHRP: sodium azide, cyanide, L-cystine, dichromate, ethylenethiourea,hydroxylamine, sulfide, vanadate, p-aminobenzoic acid, Cd⁺², Co⁺², Cu⁺²,Fe⁺³, Mn⁺², Ni⁺², Pb⁺³. Many stop reagents known to the art make use ofthese compounds. Another known reagent used to stop the HRP activity isoxalic acid.

When designing a stop reagent for reactions catalyzed by HRP, bleachingof the color brought about by the chromogenic substance is a frequentproblem. The use of heavy metal salts in stop reagents has a number ofdisadvantages including toxicity. Also, certain salts of heavy metalsare explosive as dry materials. Another known stop reagent for the HRPreaction is formaldehyde. However, the stabilizing effect of thiscompound is unsatisfactory. In addition, formaldehyde is toxic and has atroublesome smell.

U.S. Pat. No. 4,234,680 teaches the use of an alkali metal bisulphite asstop reagent. However, this reducing agent has the potential ofdecolorizing the oxidized products formed from certain chromogenicsubstrates by the HRP catalyzed reaction. This applies especially to theoxidized products of ABTS and related salts.

In U.S. Pat. No. 4,752,570 a process for the determination of peroxidaseis described, wherein a chromogenic substrate reaction is stopped byadding catalase as stop reagent. This process, however, requires the useof an enzyme with limited stability and causes foaming due to therelease of oxygen. Foaming may interfere with spectrophotometricreadings.

Finally, surface-active agents, such as secondary alkyl sulphate ordodecyl hydrogen sulphate, have been suggested as stop agents. Forexample, SDS (sodium dodecyl sulphate) at a final concentration insolution of 0.5% [w/v] is suggested for stopping the color formationusing HRP and ABTS, see pack insert of product #11684302 (catalogue ofRoche Diagnostics GmbH, Mannheim, Germany). Similarly, an SDS solutionis supplied from KPL laboratories for the same purpose. However, thesecompounds are not able to fully suppress further color formation and/orlead to precipitation of the substrate. Results presented in Example 1illustrates this fact.

Also strong (mineral) acids are used as stop reagents to inhibitperoxidase enzymatic activity. This is especially the case when3,3′-5,5′-tetramethylbenzidine serves as a color substrate and theperoxidase enzyme is inhibited by adjusting the pH to values of pH 2 oreven lower.

Several reagents that are described above lead to precipitation ofcolored substrates like ABTS and cause erroneous readings, e.g.spectrophotometric readings of ELISA plates. Precipitation particularlyoccurs when using strong acids at pH values lower than 2. In addition, 1M H₂So₄ changes the color of the substrate3,3′-5,5′-tetramethylbenzidine from blue to yellow. Other inhibitors(e.g. bisulphate, see above) may lead to bleaching and therefore are oflimited use. Furthermore, certain stop reagents only after a certain lagphase achieve inhibition or just lead to an incomplete inhibition of theHRP-catalyzed reaction.

It is an object of the present invention to overcome the disadvantagesof the stop reagents of the state of the art. It is a particular objectof the present invention to provide an improved stop reagent forHRP-catalyzed reactions. Another object of the invention is to provide aprocess for the determination of peroxidase enzymatic activity in asample.

SUMMARY OF THE INVENTION

A first embodiment of the invention is the use of an acidic aqueoussolution of a protein denaturing agent to inhibit peroxidase enzymaticactivity. A further embodiment of the invention is a compositioncomprising (a) a peroxidase enzyme, (b) an electron acceptor substrate,(c) an electron donor substrate, and (d) a protein denaturing agent,characterized in that the composition is an acidic aqueous solution.Yet, a further embodiment of the invention is a method to inhibitperoxidase enzymatic activity in an aqueous solution comprising aperoxidase enzyme, an electron acceptor substrate, and an electron donorsubstrate, characterized in that the composition is mixed with an acidicaqueous solution of a denaturing agent, whereby in the resulting mixturethe pH is adjusted to a final value of pH 2.5 to pH 4.2. Yet, a furtherembodiment of the invention is a kit comprising (a) a conjugatecomprising horseradish peroxidase, (b) an acidic solution containing aprotein denaturing agent. Yet, a further embodiment of the invention isa method to determine the amount of peroxidase enzymatic activity in asample, comprising the steps of (a) adding to the sample an electronacceptor substrate and an electron donor substrate, whereby the electrondonor substrate forms a dye or a pigment upon oxidation; (b) incubatingthe sample, whereby the peroxidase enzymatic activity catalyzes theoxidation of the electron donor substrate; (c) inhibiting the peroxidaseenzymatic activity by way of mixing with the sample an acidic aqueoussolution of a protein denaturing agent, whereby the pH value of themixture is adjusted at an acidic pH; (d) determining the amount of dyeor pigment formed; (e) correlating the amount of dye or pigment with theamount of peroxidase enzymatic activity in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, A and B: inhibition of ABTS turnover catalyzed by a low amountof HRP (detection of a low amount of mono- and oligosomes in Jurkatcells; further details are given in Example 1). The ordinate indicatesabsorbance. Groups of bars represent comparative measurements at timepoints after the addition of stop reagents as indicated: (‡)—control,ABTS solution incubated without HRP, no stop reagent added; (a) to(k)—ABTS solution incubated with HRP and subsequently mixed with stopreagent as indicated: (a) 1% SDS, (b) 5% SDS, (c) distilled water, (d)0.5 M H₂SO₄, (e) 0.25 M H₂SO₄, (f) 0.1 M H₂SO₄, (g) 0.05 M H₂SO₄, (h)0.25 M oxalic acid, (i) 0.1 M oxalic acid, (k) 0.05 M oxalic acid, (l)distilled water.

FIG. 2, A and B: inhibition of ABTS turnover catalyzed by a high amountof HRP detection of a high amount of mono- and oligosomes in Jurkatcells; further details are given in Example 1. All other designationsare the same as in FIG. 1.

DETAILED DESCRIPTION OF THE INVENTION

Certain terms are used with particular meaning, or are defined for thefirst time, in this description of the present invention. For thepurposes of the present invention, the terms are defined by theirart-accepted definitions, when such exist, except that when thosedefinitions conflict or partially conflict with the definitions setforth below. In the event of a conflict in definition, the meaning ofthe terms are first defined by the definitions set forth below.

The term “comprising” is used in the description of the invention and inthe claims to mean “including, but not necessarily limited to.”

Horseradish peroxidase is also referred to as “HRP.” Generally, themixture of horseradish peroxidase isoenzymes isolated from horseradishroots is within the meaning of the term HRP. However, the term HRP alsoencompasses enriched, isolated, or recombinantly produced HRP Cisoenzyme. Peroxidase (particularly HRP) activity can be assayed in a“reaction mixture” which comprises an aqueous solution (a) a peroxidaseenzyme, (b) an electron acceptor substrate, and (c) an electron donorsubstrate, whereby the salt content and the pH of the aqueous solutionpermit the peroxidase enzyme to catalyze the transfer of electrons fromthe electron donor substrate to the electron acceptor substrate.Regarding the electron donor substrate the invention generallycontemplates compounds which upon oxidation form a dye or a pigment.Thus, the invention encompasses stop reagents for peroxidase-catalyzedreactions using (1) chromogenic, (2) fluorogenic, and (3) light emittingelectron donor substrates. Such substrates are well known to the art.

As used herein, the term “stop reagent” refers to a reagent forterminating or inhibiting in a reaction mixture the chemical reactioncatalyzed by HRP. In addition, the stop reagent stabilizes the chemicalentities formed by the chemical reaction catalyzed by HRP, so that theamount of oxidized reaction product(s) of the reaction can be determinedfollowing the addition of the stop reagent to the reaction mixture. Itis understood that the stop reagent according to the invention itselfusually comprises a mixture of different compounds.

According to the present invention, a “protein denaturing agent” or“denaturing agent” is a chemical compound capable of denaturing proteinsin aqueous solution. Denaturation is usually effected by changing thesecondary or tertiary structure of the protein, particularly HRP. Thus,certain detergents are capable of changing the structure of HRP. Thesame result can be obtained by changing the interaction of the protein(HRP) with the solvent (water), e.g. by way of adding chaotropicsubstances. A high concentration of a chaotropic substance changes thebulk properties of water (Cacace, M. G., et al., Quarterly Review ofBiophysics 30 (1997) 241-277) by weakening hydrophobic interactions,thereby causing proteins to denature. Whether effected by a detergent ora chaotropic substance, denaturation is not necessarily quantitative.Denaturation may be dependent, e.g., on the concentration of the proteindenaturing agent.

A first embodiment of the invention is the use of an acidic aqueoussolution of a protein denaturing agent as a stop reagent to inhibitperoxidase enzymatic activity. As the skilled person appreciates, thestop reagent of the present invention is capable of stopping the colorformation in a peroxidase reaction mixture at a definite, predeterminedpoint of time. The stop reagent of the invention is immediatelyeffective and does not result in a disadvantageous change of the colorpresent at the point of time of its addition to the reaction mixture.

The stop reagent of the invention comprises at least two essentialcomponents, wherein the first essential component shifts the pH of thereaction mixture to a value between 2.5 and 4.2, but without leading tothe precipitation of the reaction product. That is to say, the reactionmixture is acidified mildly thereby avoiding precipitation of any memberof the reaction mixture. According to the invention an aqueous solutioncomprising a protein denaturing agent is used to inhibit peroxidaseenzymatic activity, whereby, the pH of the aqueous solution ispreferably between pH 2.5 and pH 4.2. More preferred, the pH is between2.7 and 3.8, even more preferred, between pH 3 and pH 3.5. Anotherpreferred pH range of the aqueous solution is between pH 2.5 and pH 3.Yet another preferred pH range of the aqueous solution is between pH 3and pH 4.2.

The second essential component of the stop reagent is a proteindenaturing agent which preferably is a detergent or a chaotropicsubstance. A preferred detergent is selected from the group consistingof SDS (sodium dodecylsulfate), N-Laurylsarcosin, cetyltrimethylammoniumbromide (CTAB), and dodecyltrimethylammonium bromide (DTAB) as well asmixtures thereof. A preferred chaotropic substance is selected from thegroup consisting of urea or guanidinium hydrochloride, guanidiniumisothioocyanate, and guanidinium thiocyanate, as well as mixturesthereof. Taken alone each of the said protein denaturing agents will notlead to an immediate and complete inhibition at the concentrationapplied. The surprising finding by the inventors was, however, that thecombination of any one of the protein denaturants combined with anacidic pH leads to an exceptionally effective inhibition of peroxidaseenzymatic activity.

Another embodiment of the invention is a method to inhibit peroxidaseenzymatic activity in an aqueous solution comprising a peroxidaseenzyme, an electron acceptor substrate, and an electron donor substrate,characterized in that the composition is mixed with an acidic aqueoussolution of a denaturing agent, whereby in the resulting mixture the pHis adjusted to a final value of pH 2.5 to pH 4.2. It is preferred thatthe final concentration of the denaturing agent is 0.25 to 10% weight byvolume when the denaturing agent is a detergent. In case the denaturingagent is a chaotropic substance the preferred concentration is 5% to 70%weight by volume. In order to achieve very good HRP inactivation, it isvery much preferred to inhibit peroxidase enzymatic activity in thereaction mixture with SDS at a final concentration between 0.25% and 10%weight by volume, most preferred between 0.5% and 2.5% weight by volume.Thus, applying the method of the invention requires the skilled personto form a composition of the invention, the composition comprising (a) aperoxidase enzyme, (b) an electron acceptor substrate, (c) an electrondonor substrate, and (d) a protein denaturing agent, characterized inthat the composition is an acidic aqueous solution.

In a preferred embodiment, the electron donor substrate is achromogenic, chemiluminescent or fluorgenic compound. The stop solutionof the present invention can be used with the electron donor substratesknown to the art for carrying out HRP determinations. Typical examplesof frequently used electron donor substrates include2,2′-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt(ABTS), 3,3′-5,5′-tetramethylbenzidine (TMB), 4-hydroxy-phenethylamine(tyramine), 4-hydroxyphenyl acetic acid, homovanillic acid,o-phenylenediamine, p-phenylenediamine, m-aminosalicylic acid,dianisidine, p-aminobenzoic acid, aniline, 4-aminoantipyrine and thelike. These chromogens are well known for HRP determinations and do nothere require any further explanation.

Usually the electron acceptor substrate is hydrogen peroxide. However,other electron acceptor substrates are possible.

The present invention also provides a process for the determination ofperoxidase. An example therefor is an enzyme immunoassay in which animmobilized analyte is qualitatively or quantitatively detected by meansof an antibody or antibody fragment conjugated to one or more peroxidaseenzymes. In such a case the amount of bound peroxidase is proportionalto the amount of analyte. Such an assay requires reacting the peroxidasewith a peroxide and a substrate, and kinetic or end point measurement ofthe color resulting from the oxidation of the chromogen. In such assayscolor formation needs to be stopped after a definite period of time bythe addition of a stop agent. Thus, a further embodiment of theinvention is a method to determine the amount of peroxidase enzymaticactivity in a sample, comprising the steps of (a) adding to the samplean electron acceptor substrate and an electron donor substrate, wherebythe electron donor substrate forms a dye or a pigment upon oxidation;(b) incubating the sample, whereby the peroxidase enzymatic activitycatalyzes the oxidation of the electron donor substrate; (c) inhibitingthe peroxidase enzymatic activity by way of adding to the sample anacidic aqueous solution of a protein denaturing agent; (d) determiningthe amount of dye or pigment formed; (e) correlating the amount of dyeor pigment with the amount of peroxidase enzymatic activity in thesample.

A further embodiment of the invention is a kit comprising (a) aconjugate comprising horseradish peroxidase, (b) an acidic solutioncontaining a protein denaturing agent. A preferred kit, according to theinvention additionally comprises one or more microwell plates, achromogenic, fluorogenic, or light emitting electron donor substrate,and an electron acceptor substrate. Also preferred, a kit containsperoxidase enzyme or an analyte binding agent (such as an antibody)conjugated to one or more peroxidase enzymes.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

SPECIFIC EMBODIMENTS Example 1 Inhibition of High and Low Amounts of HRPActivity Using SDS, Sulfuric Acid, and Oxalic Acid Stop Reagents

Jurkat cells were cultured and subsequently divided into two culturedishes. In one dish the cells were treated with 1 μg/ml CAM(Camptothecin) to induce apoptosis, thereby leading to the formation ofmono and oligonucleosomes, the target antigen. After an incubationperiod of 4 hours the cells of both dishes were harvested separately bycentrifugation, lysed by the detergent contained in the Cell DeathDetection ELISA Plus (Roche Diagnostics GmbH, Mannheim, Germany; CatalogNo. 1920685), and frozen. The mono and oligonucleosomes consisting ofDNA and histones H2A, H2B, H3 and H4 were detected in thawed cellhomogenate by way of a sandwich ELISA. Antigen was captured with a firstmonoclonal antibody against histone from the Cell Death Detection ELISAPlus kit. The antibody was coated onto the walls of a 96-wellmicroplate. The detection antibody was a DNA-specific polyclonalantibody conjugated with horseradish peroxidase.

Non-induced and CAM-induced Jurkat cells yielded low and high amounts oftarget antigen, thereby leading to low and high absolute amounts ofhorseradish peroxidase enzymatic activity in the respective wells.

Into each well of the 96-well microplate an aliquot of 100 μl of ABTSSolution (Roche Diagnostics GmbH, Mannheim, Germany; Roche AppliedScience, product no. 11684302001) was added. After an incubation for 8minutes at room temperature, 100 μl of either distilled water or a stopreagent as indicated in the legend to FIG. 1 was added. Absorbance wasmeasured at 405 nm at the time points indicated in FIG. 1.

As can be seen in FIG. 1, SDS and high concentrations of oxalic acid orsulfuric acid lead to a complete inhibition of the reaction. In FIG. 2,however, it becomes clear that SDS and oxalic acid were not able toinhibit completely the high amount of HRP activity. In addition, H₂SO₄and oxalic acid at high concentrations of 0.5 to 0.25M causeprecipitation of the substrate.

Example 2 Inhibition of 1 mU of HRP Activity Using Heavy Metal Salts andOther Compounds as Stop Reagents

Various described peroxidase inhibitor compounds as given in Table 1were tested as stop reagents at the indicated concentrations with HRP in100 μl of ABTS solution in a 96-well plate using a differentexperimental setting: HRP was added to the ABTS solution at an amount toyield an absolute peroxidase enzymatic activity of 1 mU per well. Stopreagent was added following an initial incubation of the ABTS/HRPmixture for 8 minutes at room temperature. For each stop reagent,several photometric readings were made after the time spans indicated inTable 2a and Table 2b. In these tables typical results of inhibitionassays with the inhibitors of Table 1 are shown. When two differentconcentrations of an inhibitor were tested, only the result obtainedwith the higher concentration of the inhibitor was included in thetables. At the lower concentrations the inhibitors were not sufficientlyeffective when tested (data not shown).

TABLE 1 Inhibitors of HRP tested Inhibitor 1st concentration 2ndconcentration (i) Manganese(II) carbonate 0.25 mM (ii) Lead(II) acetate0.5M (iii) Cobalt(II) acetate 0.5M 0.05M (iv) Cadmium(II) sulfate 0.5M0.05M (v) Iron(III) nitrate 0.5M (vi) Copper(II) sulfate 0.5M 0.05M(vii) Sodium orthovanadate 0.5M 0.05M (viii) NaF 0.4M (ix) Nickel (II)chloride 0.1M (x) Sodium azide 0.25M 0.025M (xi) Sodium Oxamate 0.1M(xii) KSCN 0.1M 0.05M (xiii) NaOH 0.5M 0.1M

It was also noted that vanadate, sodium hydroxide, potassium thiocyanateand sodium azide resulted in a decoloration of the dye, whereas only NaFand the commercially available stop reagent (designated “comm.,” inTable 2b) affected inhibition, however only partially.

TABLE 2a Absorbance at 405 nm after time intervals as indicated Time ‡(i) (ii) (iii) (iv) (v) (xiii) (vi) 6 min 1.090 1.066 1.065 1.062 1.0591.054 1.036 1.077 9 min 1.413 1.560 1.864 1.471 1.759 3.999 0.878 1.64615 min 2.183 2.173 0.761 2.666 2.907 3.999 0.027 1.669 30 min 3.2553.260 1.900 2.666 3.999 3.999 0.027 1.767 45 min 3.565 3.767 2.998 2.6663.999 3.999 0.027 1.831 60 min 3.763 3.999 3.999 2.666 3.999 3.999 0.0271.765 180 min 3.999 3.999 3.999 n.d. [*] 2.379 3.999 0.026 1.842 ‡indicates the control: ABTS solution incubated without HRP, no stopreagent added. [*] negative value deleted.

TABLE 2b Absorbance at 405 nm after time intervals as indicated Time ‡(vii) (viii) (ix) (x) (xi) (xii) comm. 6 min 1.189 1.173 1.176 1.1751.172 1.182 1.179 1.159 9 min 1.531 0.115 1.343 2.241 1.395 1.776 1.2071.948 15 min 2.459 0.096 1.424 2.999 0.111 2.525 1.290 2.453 30 min3.999 0.089 1.572 3.719 0.028 3.091 1.201 2.699 45 min 3.999 0.088 1.7213.999 0.026 3.097 1.086 2.704 60 min 3.999 0.092 1.818 3.715 0.024 3.0950.989 2.693 180 min 3.999 0.100 2.107 3.999 0.024 2.865 0.849 2.634 ‡indicates the control: ABTS solution incubated without HRP, no stopreagent added. “comm.” Indicates a commercially available stop reagentfrom KPL, Inc., Gaithersburg, MD, USA; catalogue no. 50-85-01

Example 3 Inhibition of 1 mU of HRP Activity Using Acidified SDSSolution as Stop Reagent

A solution of 5% [w/v] sodium dodecyl sulfate acidified with 0.25 Mhydrochloric acid was used as a stop reagent to inhibit 1 mU peroxidaseenzymatic activity of HRP. The experimental setting was similar to thatin Example 2, using an amount of 1 mU of HRP activity in 100 μl of ABTSsolution. 100 μl of stop reagent were added.

TABLE 3 Absorbance at 405 nm after time intervals as indicated ‡(control) 5% SDS/25 mM HCl  5 min 1.134 1.157  7 min 1.378 1.866 10 min1.824 1.853 15 min 2.902 1.841 30 min 3.999 1.833 45 min 3.999 1.833 60min 3.999 1.837 120 min  3.999 1.845 (‡) indicates the control: ABTSsolution incubated without HRP, no stop reagent added.

Another experiment was performed in which two different SDSconcentrations, 5% and 0.5% [w/v] in an acidified solution werecompared.

TABLE 4 Absorbance at 405 nm after time intervals as indicated ‡(control) 5% SDS/25 mM HCl 0.5% SDS/25 mM HCl  5 min 0.848 0.850 0.846 7 min 1.122 1.288 1.178 10 min 1.703 1.283 1.219 15 min 2.507 1.2841.246 30 min 3.999 1.281 1.259 45 min 3.745 1.273 1.250 60 min 3.7591.272 1.246

It was found that acidification of the reaction mixture in the well to apH value of about 2.5 to 4.2 in combination with SDS at a finalconcentration of between 0.25% and 2.5% weight by volume is veryeffective in inhibiting peroxidase enzymatic activity. At the same time,the concentration of the acidic component does not cause precipitationof the substrate.

What is claimed is:
 1. A method for determining the amount of peroxidaseenzymatic activity in a sample comprising a peroxidase enzyme, saidmethod comprising the steps of: providing an aqueous solution comprisingsaid sample, an electron acceptor substrate, and an electron donorsubstrate comprising ABTS(2,2′-azino-di-(3-ethylbenzothiazoline-6-sulphonic acid), wherein theABTS forms a chromogenic product upon oxidation catalyzed by theperoxidase enzyme, inhibiting the peroxidase enzymatic activity whilestabilizing the chromogenic product by adding a acidic stop reagentcomprising an amount of sodium dodecyl sulfate (SDS) sufficient toprovide a solution concentration of SDS between 0.25% and 2.5% weight byvolume, whereby the pH of the solution is adjusted to between about 2.5and 4.2, determining the amount of chromogenic product formed, andcorrelating the amount of chromogenic product formed with the amount ofperoxidase enzymatic activity in the sample.
 2. The method of claim 1wherein the stop reagent further comprises a chaotropic substanceselected from the group consisting of urea, guanidinium hydrochloride,guanidinium thiocyanate, and guanidinium isothiocyanate.
 3. The methodof claim 2 wherein the chaotropic substance has a concentration of 5% to70% weight by volume.
 4. The method according to claim 1, wherein theacidic stop reagent comprises hydrochloric acid (HCl).
 5. The methodaccording to claim 4, wherein the acidic stop reagent comprises 0.25 MHCl.
 6. The method according to claim 1, wherein the electron acceptorsubstrate comprises H202.
 7. A method for determining the amount ofperoxidase enzymatic activity in a sample comprising a peroxidaseenzyme, said method comprising the steps of: providing an aqueoussolution comprising said sample, an electron acceptor substrate, and anelectron donor substrate, wherein the electron donor substrate forms achromogenic product upon oxidation catalyzed by the peroxidase enzyme,inhibiting the peroxidase enzymatic activity while stabilizing thechromogenic product by adding a acidic stop reagent comprising an amountof sodium dodecyl sulfate (SDS) sufficient to provide a solutionconcentration of SDS between 0.25% and 2.5% weight by volume, wherebythe pH of the solution is adjusted to between about 2.5 and 4.2,determining the amount of chromogenic product formed, and correlatingthe amount of chromogenic product formed with the amount of peroxidaseenzymatic activity in the sample.